1. Field of the Invention
The present invention relates to selenium compounds, and more specifically to selenium compounds which, when covalently attached to functional and site directed molecules and devices, produce superoxide and other reactive compounds in the presence of thiols.
2. Description of the Invention Background
Selenium (Se) is among the most toxic of all known minerals. Its toxicity symptoms in horses were most likely described by Marco Polo while traveling the silk road in China. In the 1920's, loss of livestock in parts of the western and central United States was severe. Those losses of livestock were investigated by the United States Department of Agriculture Experiment Station in South Dakota. In 1934, the cause of the loss of livestock was traced by the Experiment Station to the element selenium which was high in certain soils and high secondarily in plants from several species of Astragalus (vetch), Xylorrhiza (woody aster), conopsis (goldenrod) and Stanleya (Prince's Plume). Ingestion of these and other Se containing plants by livestock often proved to be fatal.
Throughout the period of time between the discovery of selenium toxicity in livestock in 1934 and 1988, many hypotheses were put forth to explain the mechanism by which many but not all compounds of selenium were toxic. None of these theories of selenium toxicity proved satisfactory in fully explaining why selenium was toxic. In 1989, Seko, Y. E., Saito, Y. Kitahare, J. and Imura, N., "Active oxygen generation by the reaction of Selenite with reduced glutathione in vitro," In: Proceedings of the fourth international symposium on selenium and medicine (ed., Wendel, A.) pp.70-73, Springer-Verlag, Heidelberg, Germany, (1989), reported that selenite, (SeO.sub.3), an inorganic form of Se, reacted with a thiol, glutathione, (GSH), to produce superoxide (O.sub.2..sup.-). Since superoxide is a known toxicant, this raised the possibility that all selenium compounds that are toxic might generate superoxide. Through the testing of many selenium compounds, it was found that the inorganic compounds SeO.sub.3 and selenium dioxide (SeO.sub.2) were able to generate O.sub.2..sup.- and hydrogen peroxide (H.sub.2 O.sub.2) when presented with a thiol, such as glutathione, cysteine (CysSH), or dithiothreitol D(SH).sub.2. Furthermore, it was found that all diselenides of the composition RSeSeR tested likewise would generate O.sub.2..sup.- and H.sub.2 O.sub.2 when presented with any of the before mentioned thiols.
In 1947, Feigl, F. and West, P. W. "Test for selenium based on a catalytic effect", Analytical Chemistry, vol.19, pp.351-353 (1947), reported that selenium could catalyze a redox reaction involving sulfide oxidation. This soon became a common test for selenium using methylene blue. This reaction was further studied by others using different selenium compounds and thiols, demonstrating catalysis for some but not all selenium compounds. See, West, P. W. and Ramakrishna, T. V. "A catalytic method for determining traces of selenium," Analytic Chemistry, vol.40, pp.966-968 (1968); Levander, O. A., Morris V. C., and Higgs, D. J. "Selenium as a catalyst for the reduction of cytochrome C by glutathione", Biochemistry, vol.12, pp.4591-4595 (1973), Rhead, N. J. and Schrauzer, G. N., "The selenium catalyzed reduction of methylene blue by thiols", Biorganic Chemistry, vol.3, pp.225-242 (1974). The selenium catalytic activity of selenocystine (RSeSeR) in the presence of thiols was reported in 1958. It is now believed that all of the foregoing reactions of selenium compounds produce superoxide. See, Xu, M., Zhang, L., Sun, E. and Fan, H., "Studies on the interaction of trace element selenium with oxygen free radical," Advances in Free Radical Biology and Medicine, vol.1, pp.35-48 (1991); Xu, H., Feng, Z., and Yi, C., "Free radical mechanism of the toxicity of selenium compounds," Huzahong Longong Daxus Xuebao, vol.19, pp.13-19 (1991); Kitahara, J., Seko, Y. and Imura, N., "Possible involvement of active oxygen species in selenite. toxicity in isolated rat hepatocytes," Archives of toxicology, vol.67, pp.497-501 (1993); Chaudiere, J., Courtin, O. and Ledaire, J., "Glutathione oxidase activity of seleno-cystamine: a mechanistic study," Archives of Biochemistry and Biophysics, vol.296, pp.328-336 (1992).
Selenium and a number of its compounds have been known since the early 1970's to possess anti-cancer properties. It has been generally recognized that selenite and selenium dioxide are good anti-cancer agents in vitro and in experimental animals and that the compounds are also cytotoxic to both cancer and normal cells in vitro. U.S. Pat. No. 5,104,852 to Kralick et al. describes the use of selenodiglutathione and other selenodithiols of the configuration (GSSeSG) to treat cancer. Selenodiglutathione is the product of the reaction between selenite or selenium dioxide with glutathione. The compound, selenodiglutathione, has been isolated. U.S. Pat. No. 5,104,852, however, does not describe the mechanism of action by which selenodiglutathione and like compounds are useful in treating cancer.
In 1982, the interaction of selenite and selenocystine with glutathione in the cytotoxicity and lysis of rat erythrocyte membranes was described by Hu, M. L. and Spallholz, J. E., "In vitro hemolysis of rat erythrocytes by selenium compounds", Biochemical Pharmacology, vol.32, pp.857-961 (1983). This cytotoxicity, as revealed by scanning electron microscopy of rat erythrocytes, caused the erythrocyte membranes to become burred, the cells to quadruple in size and lyse similar to that described by Kellogg, E. and Fridovich, I., "Liposome oxidation and erythrocyte lysis by enzymically generated superoxide and hydrogen peroxide," J. Biol. Chem., vol.252, pp.6721-6728 (1977). This toxicity, however, was not expressed by selenomethionine, a compound possessing the configuration RSeR. In 1991, an article by Yan, L. and Spallholz, J. E., "Free radical generation by selenium compounds," FASEB J., vol.5, p.A581 (1991), showed a dose responsive toxicity of several selenium compounds to a human mammary tumor cell line. Additional investigations using lucigenin chemiluminescence and luminol chemiluminescence revealed a dose response in O.sub.2..sup.- and H.sub.2 O.sub.2 generated chemiluminescence by selenite, selenium dioxide and all selenium compounds tested of the configuration RSeSeR. It was found furthermore, that selenium compounds in the presence of either tumor cells or glutathione alone produced superoxide and H.sub.2 O.sub.2. Chemiluminescence from the reactions of lucigenin with O.sub.2..sup.- or luminol with H.sub.2 O.sub.2 could be quenched by the native enzymes superoxide dismutase (SOD), catalase (CT) or glutathione peroxidase (GSHPx). Denatured enzymes would not quench these reactions, confirming the generation of the free radical (O.sub.2..sup.-) and H.sub.2 O.sub.2 by selenium compounds and thiols. All of this selenium free radical chemistry has been reviewed by Spallholz, J. E., "On the nature of selenium toxicity and carcinostatic activity," Free Radical Biology and Medicine, vol.17, pp.45-64, (1994).
A summation of this large body of experimental data on selenium toxicity, catalysis and carcinostatic activity is as follows:
1) The selenium compounds, SeO.sub.2 and SeO.sub.3, react with thiols to produce a selenodithiol of the configuration (RSSeSR). This compound is not toxic per se nor is it carcinostatic. The toxic carcinostatic form of RSeR is the reduced selenide anion, RSe.sup.-. This selenopersulfide form of Se is catalytic as shown by the inhibition of both catalysis and superoxide generation by iodoacetic acid and mercaptosuccinic acid. PA1 2) Selenium compounds of the configuration (RSeSeR) or (RSeSeR') react with thiols to produce the reduced selenite anion RSe.sup.- or R'Se.sup.-. This selenopersulfide form of Se is catalytic as shown by the inhibition of both catalysis and superoxide generation by iodoacetic acid and mercaptosuccinic acid. PA1 3) Organic selenium catalysts by the configuration RSe.sup.- the selenopersulfide anion, is catalytic in the presence of thiols and RSe.sup.- continues to generate superoxide (O.sub.2..sup.-) ion as long as sufficient concentrations of O.sub.2..sup.- and thiol are in the medium. Selenium compounds derived from selenite or selenium dioxide reacting with glutathione (GSH) are converted to elemental selenium (Se.) follows; Seo.sub.3 (SeO.sub.2)+2GSH-2GSSeSG-2GSSG +Se.. Elemental selenium (Se.) is non-catalytic and not toxic. PA1 4) Compounds of selenium of the configuration RSe.sup.- are toxic due to the catalytic acceleration of thiol oxidation which produces O.sub.2..sup.-, H.sub.2 O.sub.2 and the more toxic free radical, the hydroxyl radical (.OH). This chemistry had been discussed by Misra, H. P., "Generation of superoxide free radical during the auto-oxidation of thiols," J. Biol. Chem., vol.249, pp.2151-2155 (1974) for the spontaneous oxidation of thiols. The association of rapid thiol catalysis by selenium compounds of the configuration RSe.sup.- and the toxicity from which it produced free radicals and reactive toxic oxygen products was recognized in 1992 by one of the inventors.
The use of selenium for the treatment of experimental cancer in animals and cancer in humans in vivo has been extensively described by many authors, such as Milner, J. A., Greeder, G. A., Poirier, K. A., "Selenium and transplantable tumors," (Spallholz, J. E., Martin, J. L., Ganther, H. E., eds.) Selenium in Biology and Medicine, AVI Publishing Co. (1981); Ip, C. and Ganther, J. E., "Relationship between the chemical form of selenium and anticarcinogenic activity," CRC Press, Inc., pp.479-488 (1992); Caffrey, P. B. and Frenkel, G. D. "Selenite cytotoxicity in drug resistant and nonresistant human ovarian tumor cells," Cancer Research, vol.52, pp.4812-4816 (1992); Schrauzer, G. N., "Selenium: Mechanistic aspects of anticarcinogen action", Biol. Trace Elem. Res., vol.33, pp.51-62 (1992); Yan, L. and Spallholz, J. E. "Generation of reactive oxygen species from the reaction of selenium compounds with thiols and mammary tumor cells," Biochemical Pharmacology, vol.45, pp.429-437 (1993). The use of selenium as a cytotoxic agent to both normal cells and cancer cells in vitro has been described in U.S. Pat. No. 5,104,852 for the injection of selenodiglutathione into a tumor mass to kill tumor cells. In U.S. Pat. No. 4,671,958, Rodwell et al. described many antibacterial drugs, 3 antiviral drugs, 1 antifungal drug, 7 antineoplastic drugs, 3 radiopharmaceuticals, 3 heavy metals and 2 antimycoplasmals as drugs for antibody mediated delivery. The pharmacology for all of these drugs which are listed in Table 1 of U.S. Pat. No. 4,671,958 is generally understood. Table 1 of the Rodwell et al. patent does not contain selenium because its pharmacological action as a free radical generator of (O.sub.2..sup.-) and other reactive oxygen molecules was not understood or known at that time.
Viral infections are difficult to treat because viruses lack an uptake mechanism by which agents designed to kill or damage the virus can be taken into the virus. Even when it is known that a person has been infected with a virus, it has been necessary heretofore to wait for the virus to infect cells in the host and reproduce in the cells. The cell, which does have an uptake mechanism, will takeup the agent. Viral reproduction can only then be blocked. In practice, the viral infection has usually spread by the time treatment is possible. This is particularly devastating with HIV viral infections, the causative agent for acquired immune deficiency syndrome (AIDS). Often, an infected person knows that he or she has been exposed to the virus but is unable to stop the spread of the virus until that person's cells have been infected.
Once-reliable antibiotics for treating bacterial infections are falling by the way side. With the emergence of many conventional drug-resistant strains of bacteria, such as those identified in the Mar. 28, 1994 issue of Newsweek magazine and set forth below in Table 1, the need for a new agent for combating infectious diseases is becoming critical.
TABLE 1 ______________________________________ DRUG RESISTANT BACTERIA ANTIBIOTICS THAT BACTERIA DISEASES CAUSED NO LONGER WORK ______________________________________ Enterococcus Blood poisoning, Aminoglycosides, surgical infections cephalosporins, erythromycin, penicillin, tetracycline, vancomycin Hemophilus Meningitis, ear Chloramphenicol, influenza infections, penicillins, pneumonia, tetracycline, sinusitis trimethorprim/ sulfamethoxazola Mycobacterium Tuberculosis Aminoglycosides, tuberculosis ethambutol, isoniazid, pyrazinamide, rifampin Neisseria Gonorrhea Penicillins, gonorrhea spectinomycin, tetracycline Plasmodium Malaria Chloroquine falciparum Shigella Severe Diarrhea Ampicillin, dysenteriae chloramphenicol, tetracycline, trimethoprim/ sulfamethoxazole Staphylococcus Blood poisoning, All but aureus pneumonia, surgical Vancomycin infections Streptococcus Meningitis, Aminoglycosides, pneumoniae pneumonia cephalosporins, chloramphenicol, crythromycin, penicillins, tetracycline, trimethorprim/ sulfamethoxazola ______________________________________
It is an object of the invention to provide a new bacterialcidal and viralcidal agent.
It is a further object of this invention to provide a methodology to use of the aforementioned free radical technology as bacterialcidal or viralcidal agents. It is a further object of the present invention to provide a method for directing the localized production of superoxide and descendant species thereof for selective destruction or modification of cells, tissue, membranes or extracelluler fluids to combat a variety of localized problems, from infections, to cancer, to post surgical clotting and fibrosis.